Project Summary/Abstract The assembly and trafficking of neurotropic alphaherpesviruses, including herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV), are extremely complex processes. DNA-packaged capsids must be exported from the infected cell nucleus, recruit molecular motors then move along microtubules to reach their site of envelope formation in the cytoplasm. After docking to the surface of organelles such as the trans Golgi network capsids interact with multiple viral structural proteins and the cellular ESCRT machinery to undergo envelopment. The resulting egress compartments, containing mature enveloped virions in their lumen, then recruit molecular motors and use microtubules to deliver their cargo of virions to the cell surface. Envelopment and trafficking is critical for viral infectivity and spread between cells, tissues and individuals; microtubule- directed transport is particularly dramatic in neurons, where viral particles must move very great distances within axons. Despite their importance for the spread of disease the molecular details of envelope assembly and motor recruitment are poorly understood. Central to both events is the conserved herpesvirus tegument protein UL36p (VP1/2). UL36p is essential for envelopment, and is also thought to recruit molecular motors to capsids and enveloped particles. In this proposal we explore the role of UL36p in each of these processes for both HSV-1 and PRV. Our approach combines reductionist in vitro biochemistry with fluorescence and ultrastructural microscopy, and imaging of virus assembly and movement ex vivo in living explanted neurons. To study the function of UL36p in assembly: We will explore the role of UL36p in export of capsids from the nucleus, docking to organelles and recruitment of the cellular ESCRT apparatus to the envelopment site. We also use quantitative fluorescence microscopy, in concert with correlative light and electron microscopy (CLEM), to probe the ultrastructure of HSV-1 and PRV envelopment organelles, To test the role of UL36p kinesin-binding sites in trafficking: We have identified motifs within UL36p that mediate attachment to kinesin I. We will test the importance of these binding sites for the motility of capsids and enveloped virions. To facilitate molecular and ultrastructural analysis we use an in vitro cell-free system that reconstitutes HSV-1 and PRV traffic along microtubules in a microscopic imaging chamber. In parallel we will image trafficking of naked and enveloped wild type and mutant viruses in the cell bodies and axons of sensory neuron explants cultured ex vivo.